PROJECTSUMMARY Heartvalvediseaseresultsinover23,000annualdeathsintheUnitedStateswithcalcificaorticvalvedisease (CAVD) being the most prevalent. There is no pharmacological treatment to prevent or reverse CAVD and therefore surgical intervention remains the only effective option which comes with insuperable complications andnoguaranteeoflong-termsuccess.Inhealthyindividuals,valveleafletsmaintainunidirectionalbloodflow bysustainingahighlyorganizedextracellularmatrix(ECM)structure.Themajorresidentcelltyperesponsible for ECM homeostasis is the valve interstitial cell (VIC) that resembles a fibroblast in healthy individuals. In CAVD, VICs transition towards an osteoblast-like cells and this is associated with perturbations in ECM organization including calcific nodule formation on the aortic surface leading to stenosis. Despite this, the signaling pathways underlying osteogenic changes in valve structures are not known and therefore the developmentofpharmacologicaltherapiestoattenuateorreversetheprocesshavebeenstalled. KPT-330 is a CRM1-dependent nuclear export inhibitor currently in Phase III clinical trials to treat cancer. We show that KPT-330 is sufficient to prevent CAVD in mice (Klotho-/-), as well as prevent, attenuate andrescuecalcificnoduleformationinhumanandporcineaorticVICsinvitro.Themechanismunderlyingthe beneficialeffectofKPT-330inCAVDisnotknown,butourproteomicanalysissuggeststhatitinhibitsnuclear exportoftheCRM1-dependenttranscriptionfactor,NFAT5toreducecalcification.Thegoalofthisproposalis to further test the therapeutic potential of KPT-330 in the treatment of CAVD and delineate the mechanisms underlyingitsfunction.WorkbyusandothershasshownthathumanCAVDisassociatedwithreducedSox9 and increased Wnt signaling. Interestingly, studies in chondrocytes and intestinal cells have shown that Sox9 and Wnt are commonly regulated by NFAT5, albeit in opposing directions. We have preliminary data to suggestthatKPT-330enhancescrosstalkbetweenthesesignalingpathwaysandwillthereforetesttheoverall hypothesis that: KPT-330 is a novel, therapeutic drug that treats CAVD by preventing nuclear export of NFAT5 and inhibiting osteogenic markers by increasing Sox9 and repressing Wnt. To test this we will address the following three specific aims: 1) Determine the therapeutic window of KPT-330 administration in thetreatmentofCAVDinvivo;?2)DelineatethemechanismofKPT-330mediatedtreatmentofCAVDthrough NFAT5 signaling;? and 3) Determine if NFAT5 loss of function is sufficient to cause CAVD in vivo. Upon completionwewillhavedeterminedthetherapeuticpotentialofaPhaseIIIclinicalcompound,KPT-330inthe treatment of CAVD, and delineated the mechanisms underlying its function via a novel, NFAT5-mediated signaling pathway. Together, these studies address the purpose of this basic research in CAVD RFA and findingscouldleadtoaneweffectivemedicaltherapyforthiscommondisorder.